Potential Use of Morphological Characteristics in Evaluating Natural Variation of “Barako” Seedlings

The Philippines is one of only three countries able to produce Coffea liberica and grow two other species of coffee, Coffea canephora, Coffea arabica. Coffea liberica, or “Barako,” is the least cultivated of coffee species. Its unique taste has high potential to grow in the market if production was increased. However, the 2020 Taal Volcano eruption resulted in thousands of damaged Barako trees in 2020 and 2021. As new seedlings are produced, the question of variety among planting material comes up. Farmers need a method to evaluate natural differences of current Barako trees, to answer whether seedlings from farms cultivating Barako are naturally different. This research aims to determine if the morphological characteristics of cultivated seedlings from different farms can be classified through image and statistical analysis. Basic knowledge on how varieties―which producedifferent flavors, aroma, and market value of coffee―differ among farms is needed for a strategy to increase the number of seedlings. This study’s initial data set indicated statistically significant differences in the average seedling height per node and the leaf area per length of 31, 10-month old coffee seedlings from two different farms, grown in a common environment. The process may be developed further for use in evaluating natural variation among C. liberica as seedlings

Wonder (2017): Analysis of the psychosocial dimensions of Treacher Collins´ syndrome

Anxiety and fear of the unknown are common feelings among families when children first face their schooling. But if the beginning of schooling occurs too late, the fear of social failure increases. Stephen Chbosky´s Wonder (2017) leads us to understand the complexity of this process from the perspective of Auggie and his family. Auggie is a ten-year-old boy diagnosed with Treacher Collins syndrome, who, despite having phenotypically expressed characteristics such as mandibulofacial dysostosis, has no other physical or intellectual disabilities. Having taken home schooling and belonging to a very unite family, Auggie had been the centre of attention since he was born, but now he is facing with the challenge of socialization in the school environment. Auggie will now have the opportunity to develop new and valuable skills such as kindness, empathy and companionship. In the present article we will deal with the clinical characteristics of August Pullman's protagonist character, but we will also analyse the psycho-emotional aspects that are associated with living with patients who have congenital malformations.</p

Singly Modified Amikacin and Tobramycin Derivatives Show Increased rRNA A‐Site Binding and Higher Potency against Resistant Bacteria

Semisynthetic derivatives of the clinically useful aminoglycosides tobramycin and amikacin were prepared by selectively modifying their 6'' positions with a variety of hydrogen bond donors and acceptors. Their binding to the rRNA A-site was probed using an in vitro FRET-based assay, and their antibacterial activities against several resistant strains (e.g., Pseudomonas aeruginosa, Klebsiella pneumonia, MRSA) were quantified by determining minimum inhibitory concentrations (MICs). The most potent derivatives were evaluated for their eukaryotic cytotoxicity. Most analogues displayed higher affinity for the bacterial A-site than the parent compounds. Although most tobramycin analogues exhibited no improvement in antibacterial activity, several amikacin analogues showed potent and broad-spectrum antibacterial activity against resistant bacteria. Derivatives tested for eukaryotic cytotoxicity exhibited minimal toxicity, similar to the parent compounds

Towards a Unified Taxonomy of Health Indicators: Academic Health Centers and Communities Working Together to Improve Population Health

The Clinical and Translational Science Awards (CTSA) program represents a significant public investment. To realize its major goal of improving the public's health and reducing health disparities, the CTSA Consortium's Community Engagement Key Function Committee has undertaken the challenge of developing a taxonomy of community health indicators. The objective is to initiate a unified approach for monitoring progress in improving population health outcomes. Such outcomes include, importantly, the interests and priorities of community stakeholders, plus the multiple, overlapping interests of universities and of the public health and health care professions involved in the development and use of local health care indicators. The emerging taxonomy of community health indicators that the authors propose supports alignment of CTSA activities and facilitates comparative effectiveness research across CTSAs, thereby improving the health of communities and reducing health disparities. The proposed taxonomy starts at the broadest level, determinants of health; subsequently moves to more finite categories of community health indicators; and, finally, addresses specific quantifiable measures. To illustrate the taxonomy's application, the authors have synthesized 21 health indicator projects from the literature and categorized them into international, national, or local/special jurisdictions. They furthered categorized the projects within the taxonomy by ranking indicators with the greatest representation among projects and by ranking the frequency of specific measures. They intend for the taxonomy to provide common metrics for measuring changes to population health and, thus, extend the utility of the CTSA Community Engagement Logic Model. The input of community partners will ultimately improve population health.http://journals.lww.com/academicmedicine/Abstract/publishahead/Towards_a_Unified_Taxonomy_of_Health_Indicators__.99157.asp

Anthrax edema toxin disrupts distinct steps in Rab11-dependent junctional transport

<div><p>Various bacterial toxins circumvent host defenses through overproduction of cAMP. In a previous study, we showed that edema factor (EF), an adenylate cyclase from <i>Bacillus anthracis</i>, disrupts endocytic recycling mediated by the small GTPase Rab11. As a result, cargo proteins such as cadherins fail to reach inter-cellular junctions. In the present study, we provide further mechanistic dissection of Rab11 inhibition by EF using a combination of <i>Drosophila</i> and mammalian systems. EF blocks Rab11 trafficking after the GTP-loading step, preventing a constitutively active form of Rab11 from delivering cargo vesicles to the plasma membrane. Both of the primary cAMP effector pathways -PKA and Epac/Rap1- contribute to inhibition of Rab11-mediated trafficking, but act at distinct steps of the delivery process. PKA acts early, preventing Rab11 from associating with its effectors Rip11 and Sec15. In contrast, Epac functions subsequently via the small GTPase Rap1 to block fusion of recycling endosomes with the plasma membrane, and appears to be the primary effector of EF toxicity in this process. Similarly, experiments conducted in mammalian systems reveal that Epac, but not PKA, mediates the activity of EF both in cell culture and <i>in vivo</i>. The small GTPase Arf6, which initiates endocytic retrieval of cell adhesion components, also contributes to junctional homeostasis by counteracting Rab11-dependent delivery of cargo proteins at sites of cell-cell contact. These studies have potentially significant practical implications, since chemical inhibition of either Arf6 or Epac blocks the effect of EF in cell culture and <i>in vivo</i>, opening new potential therapeutic avenues for treating symptoms caused by cAMP-inducing toxins or related barrier-disrupting pathologies.</p></div

Activation of Arf6 partially mimics the effect of EF.

<p>(A-G) Wings showing EF and Arf6-dependent phenotypes. (A) 1096GAL4>EF. (B) 1096GAL4>Arf6*. Activated Arf6 (Arf6*) causes a phenotype very similar to that caused by EF. (C) 1096GAL4>EF+Arf6*, showing an additive phenotype. (D) 1096GAL4>Arf6wt wing, displaying a wild-type phenotype. (E) 1096GAL4>EF+Arf6wt wing revealing synergy between EF and Arf6wt. (H-I) Wing imaginal discs stained with an anti Rab11 antibody. (H) In wild-type discs, Rab11 displays a dotted apical distribution. (I) In discs expressing Arf6* (1096GAL4>Arf6*), Rab11 levels are reduced, and apical restriction is lost. (J-K) Wing imaginal discs expressing Sec15-GFP. (H) Sec15-GFP expressed at high levels in 1096GAL4>Sec15-GFP discs forms large fluorescent punctae. Like EF, Arf6* expression in (K) 1096GAL4>Sec15-GFP+Arf6* discs blocks formation of Sec15-GFP punctae and reduces Sec15-GFP levels. (L-O) D-Ecad staining in wing imaginal discs. (L) D-Ecad stain in wild-type discs, revealing the apical network of AJs. (M) In 1096GAL4>Arf6* discs, apical D-Ecad levels are severely reduced, an effect similar to that of EF (N). Arf6-RNAi suppresses the EF phenotype and partially restores normal D-Ecad at AJs (O). (P) A 1096GAL4>Rab11*YFP salivary gland showing Rab11* targeting to the AJs, revealed by an anti-GFP stain. (Q) Arf6* blocks Rab11* targeting to cell junctions in 1096GAL4>Rab11*+Arf6* glands. (R) Endogenous Rab11 stain of wild-type salivary glands. (S) Arf6* causes endogenous Rab11 to accumulate in the cytoplasm and diminishes its localization at junctions. (T) D-Ecad stain of wild-type salivary glands. (U) Arf6* results in accumulation of large intracellular inclusions of D-Ecad in 1096GAL4>Arf6* salivary glands.</p

EF inhibits Rab11 downstream of GTP loading.

<p>(A-C) Wing imaginal discs expressing wild-type (wt) and mutant forms of YFP-tagged Rab11, using the strong wing-specific MS1096 GAL4 (abbreviated as 1096GAL4), and stained with a Rabbit anti-GFP antibody, reveal different sub-cellular distributions of Rab11. Insets show corresponding Z-sections. (A) Rab11wt, showing apical restriction. (B) Rab11 activated (or Rab11*), showing apical restriction plus junctional concentration, (C) Rab11 Dominant-Negative (or Rab11DN) showing loss of apical/junctional staining. The bipartite staining of Rab11* is even more pronounced in salivary glands (D-I). (D) 1096GAL4>Rab11wtYFP. (E) 1096GAL4>Rab11*YFP with higher magnification in (H). Arrows point at intercellular junctions where Rab11* accumulates. (F) 1096GAL4>Rab11*YFP +EF. EF blocks Rab11* targeting to the junctions, higher magnification shown in (I). Arrows point to intercellular junctions where Rab11* no longer accumulates when co-expressed with EF. (G) 1096GAL4>Rab11DNYFP, showing that Rab11DN does not concentrate at the junctions, but alters the morphology of secretory granules (thin arrows). (J-M): detection of the endogenous Rab11 reveals that it collects near junctions in a punctate pattern (J), with higher magnification in (L). This preference is abrogated in EF-expressing glands (K), with higher magnification in (M). Selective staining of the activated component of endogenous Rab11 (Rab11*) reveals that this functionally relevant form accumulates next to cell junctions in salivary glands (N), sagittal view in (P), an effect that is reduced in EF-expressing glands (O), sagittal view in (Q). The effect of EF on endogenous Rab11* is also visible in wing imaginal discs: (R) wt, (S) 1096GAL4>EF. Co-labeling of Rab11 and D-Ecad, the <i>Drosophila</i> ortholog of E-Cadherin (T-W) reveals co-localization of Rab11 and D-Ecad at the AJs in wt salivary glands (T), (U) higher magnification. Thick arrow indicates AJs, thin arrow indicates punctate stain near the AJs. Co-localization is lost upon EF expression (V) in salivary glands, (W) higher magnification.</p

EF prevents association between Rab11* and its effector Rip11.

<p>(A) <i>Drosophila</i> salivary glands stained with a Rabbit anti-GFP antibody (Rb α-GFP), showing that dRip11GFP accumulates at cell-cell junctions in 1096GAL4>dRip11-GFP larvae. (B) 1096GAL4>dRip11-GFP+EF glands. Junctional staining is moderately weakened by EF. (C-F) Salivary glands co-stained with a Rat anti-GFP antibody (Rt α-GFP) and a Rabbit anti-dRip11 antibody (Rb α-Rip11), showing that the co-localization between Rab11* and dRip11 detected with this antibody combination is lost or reduced upon co-expression of Rab11*YFP with EF, PKA*, or Rap1*. Lower panels show higher magnifications. Insets show representative examples of vesicles with or without Rab11*/dRip11 co-localization. (C) 1096GAL4>Rab11*YFP, arrows indicate Rab11*/dRip11 co-localization. (D) 1096GAL4>Rab11*YFP+EF. (E) 1096GAL4>Rab11*YFP+PKA*. (F) 1096GAL4>Rab11*YFP+Rap1*. Arrows point to adjacent but non-overlapping punctae. (G) Quantification of Rab11*YFP/dRip11 co-localization in panels C-F measured by the Pearson’s coefficient. (H-I) MDCK cells transfected with constructs expressing human Rip11-GFP and Rab11wt-DsRed. (H) Untreated cells showing Rab11-Rip11 co-localization throughout the cell with higher levels of Rip11 and Rab11 at cell borders. (I) Rip11 and Rab11 no longer co-localize in cells treated with ET (Edema Toxin = EF+PA). While a minor component of Rip11 is still evident at cell borders, Rab11 fails to reach the cell borders.</p

PKA* and Rap1* block Rab11 at distinct steps of junctional trafficking.

<p>(A-F) Rab11*YFP distribution (detected by a Rabbit anti-GFP antibody) is differentially affected by PKA* versus Rap1* in <i>Drosophila</i> salivary glands. (A) Rab11* (in 1096GAL4>Rab11*YFP glands) shows a strong preference for the intercellular junctions (see D for higher magnification). (B) PKA* induces ubiquitous redistribution of Rab11* (in 1096GAL4>Rab11*YFP+PKA* glands, see E for higher magnification). (C) Rap1* expression does not alter Rab11* targeting to the junctions, but blocks the final membrane fusion event (in 1096GAL4>Rab11*YFP+Rap1* glands, see F for higher magnification). (G-I) Endogenous Rab11 (detected by a mouse anti-Rab11 antibody) in salivary glands of the indicated genotypes (G) Wild-type (+/+). (H) 1096GAL4>PKA*. Rab11 shows higher levels and loss of junctional preference. (I) 1096GAL4>Rap1*, Rab11 still accumulates near the junctions. (J-L) D-Ecad staining of salivary glands. The salivary gland-specific SglGAL4 was used for these stains. (J) Linear AJs form in wild-type glands. (K) PKA* expression results in weakened junctions and cytoplasmic retention of D-ECad in punctate vesicles. (L) Rap1* expression causes D-Ecad to accumulate around the junctions in small vesicles. (M-R) Wing phenotypes implicating both cAMP effector pathways in Rab11 inhibition, from flies of the following genotypes: (M) wild-type (N) 1096GAL4>PKA*. (O) 1096GAL4>Rap1* (more similar to the EF phenotype, see panel Q). (P) 1096GAL4>Rap1*+PKA* (showing synergism between PKA* and Rap1*). (Q) 1096GAL4>EF. (R) 1096GAL4>EF+Epac^RNAi. Knock-down of Epac expression significantly suppresses the EF phenotype. See <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006603#ppat.1006603.s006" target="_blank">S6 Fig</a> showing quantifications of these phenotypes. In contrast, loss-of-function alleles of PKA-C1 do not reduce the EF phenotype significantly (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006603#ppat.1006603.s006" target="_blank">S6 Fig</a>).</p

Summary diagram.

<p>EF-induced cAMP overload can activate either or both of the PKA and Epac/Rap1 effector pathways depending on biological context. In this model, uncontrolled cAMP production by EF leads to activation of PKA and/or Epac. PKA stimulation promotes dissociation of Rab11 from its effectors Rip11 and Sec15. This premature dissociation may prevent the activated form of Rab11 from reaching the AJs, and thereby block delivery of cargo proteins (e.g., Cadherins and Notch ligands). In some cellular contexts, dissociation of Rab11-GTP from Rip11 may also lead to Rab11 degradation (e.g., <i>Drosophila</i> wing discs and human endothelial cells), while in others, only to the loss of junctional accumulation (e.g., <i>Drosophila</i> salivary glands). Over-activation of Rap1 by Epac leads to inhibition of exocyst-mediated vesicle fusion at the cell surface. Epac/Rap1 may act indirectly via sequential activation of RalA and Arf6, which cross inhibits Rab11-mediated cargo delivery to junctions, or directly through an unknown mechanism. The relative contribution of each cAMP-responsive pathway may depend on cell type and organism, although, the Epac/Rap1 branch (blocked by ESI09, and possibly by AG1024) appears to be the primary mediator of EF in fly wings, human endothelial cells, and mouse footpads. The Arf6 GTPase inhibits Rab11* targeting to the AJs, an effect that may be mediated by its interaction with exocyst components. Inhibition of Arf6 by Slit2 or Secin H3 provides protection against EF in cultured humans cells and <i>in vivo</i> in mice, respectively.</p